The processes of platelet aggregation and secretion are initiated when physiologic agonists, such as epinephrine, ADP and thrombin bind to their specific receptors on the platelet surface. In recent years, considerable information has been obtained concerning the initial binding of agonists to their receptors, and a sizeable body of information has evolved which describes the platelet's functional responses to these agonists. A relative gap exists between these two bodies of knowledge. This gap consists of the sequence of reactions that couples agonist binding to the platelet's final responses and is referred to in this proposal as signal transduction. This study will evaluate three processes involving platelet membranes that may be involved in signal transduction. First, the binding of calcium to intact platelets and to subcellular platelet membrane fractions will be evaluated. The binding of calcium to surface membranes will be related to platelet aggregation, and the association of calcium with internal membranes will be related to platelet secretion. Second, the existence of a pathway for methylation of membrane phospholipids will be explored, and the activation of this pathway by agonists will be studied. Third, the fluidity of platelet membrane lipids will be examined using fluorescent probes. Relationships will be sought between changes in membrane fluidity, phospholipid methylation, and calcium binding during platelet activation. Studies with normal platelets will be extended to platelets from individuals with congenital and acquired defects of platelet function. By clarifying the potential roles for membrane-bound calcium, lipid methylation, and membrane fluidity as intermediaries of signal transduction in human platelets, we hope to understand more fully normal platelet physiology as well as the underlying defects in some disorders of platelet function. Moreover, these studies may suggest novel ways to detect the presence of activated platelets in vitro or in vivo.